Creative Bioreactor Designs, Inc

Hybrid PGS-PCL microfibrous scaffolds with improved mechanical and biological properties

27th July 2010

Poly(glycerol sebacate) (PGS) is a biodegradable elastomer that has generated great interest as a scaffold material due to its desirable mechanical properties. However, the use of PGS in tissue engineering is limited by difficulties in casting micro- and nanofibrous structures, due to high temperatures and vacuum required for its curing and limited solubility of the cured polymer. In this paper, we developed microfibrous scaffolds made from blends of PGS and poly([epsiv]-caprolactone) (PCL) using a standard electrospinning set-up. At a given PGS:PCL ratio, higher voltage resulted in significantly smaller fibre diameters (reduced from [sim]4 µm to 2.8 µm; p < 0.05). Further increase in voltage resulted in the fusion of fibres. Similarly, higher PGS concentrations in the polymer blend resu...

Synthesis and in vivo integration of improved concentrated collagen hydrogels

26th July 2010

Normal collagen hydrogels, currently used as the dermal layer of skin substitute Apligraf®, are obtained by encapsulating dermal fibroblasts in a collagen hydrogel at low concentration (0.66 mg/ml). However they suffer from extensive contraction by cells and weak resistance against degradation, which limits their use as permanent graft. We have previously shown that concentrated collagen hydrogels at 3 mg/ml exhibit an improved performance in this respect but nevertheless degrade in vivo to ca. 50% of their initial area after 1 month. We have now investigated a new procedure to synthesize more concentrated collagen hydrogels at 5 mg/ml in order to improve hydrogel resistance and integration capability. The constructs were implanted in subcutaneous pockets in a rat model and analysed after...

A comparative study of seeding techniques and three-dimensional matrices for mesenchymal cell attachment

26th July 2010

The objective of this study was to evaluate the behaviour of MSCs during the seeding phase, using four different seeding techniques (spinner flask, custom vacuum system combined with a perfused bioreactor or with an orbital shaker, and orbital shaker) with four different scaffold materials [polyglycolic acid, poly(lactic acid), calcium phosphate and chitosan-hyaluronic acid]. Scaffolds were selected for their structural and/or chemical similarity with bone or cartilage, and characterized via scanning electron microscopy (SEM) and measurement of fluid retention. Cell attachment was compared between seeding techniques and scaffolds via cell-binding kinetics, cell viability and DNA quantification. SEM was used to evaluate cell distribution throughout the constructs. We discovered from cell su...

Engineering of polymer-based grafts with cells derived from human nucleus pulposus tissue of the lumbar spine

25th July 2010

Intervertebral disc degeneration is considered a major source of low back pain. We therefore examined an absorbable polyglycolic acid (PGA) biomaterial for its utility to support disc tissue regeneration. Microdiscectomy for lumbar disc herniation was performed in six patients. Intervertebral disc cells were isolated and in vitro cell expansion was accomplished using human serum and FGF2. In a fibrin-hyaluronan solution, disc cells were loaded on PGA scaffolds and cultured for 2 weeks. Formation of disc tissue was documented by histological staining of the extracellular matrix as well as gene expression analysis of typical disc marker genes. The use of human serum and FGF2 ensures efficient isolation and expansion of human disc cells. During this phase, dedifferentiation of the disc cells ...

Site-specific tissue inhibitor of metalloproteinase-1 governs the matrix metalloproteinases-dependent degradation of crosslinked collagen scaffolds and is correlated with interleukin-10

25th July 2010

We have previously shown that the foreign body reaction (FBR) against crosslinked collagen type I (Col-I) differs between subcutaneous and epicardial implantation sites; Col-I was quickly degraded epicardially, whereas degradation was attenuated subcutaneously. The current study set out to dissect the nature and regulation of the MMP-based degradation of implanted Col-I in mice during the FBR. Immunohistochemistry showed that MMP-2, MMP-8 and MMP-13 were present in subcutaneous and epicardial implants, whereas only MMP-9 was also present epicardially. Western blotting showed that MMP-8 and MMP-9 were mainly present in their inactive proform. In contrast, collagenase MMP-13 and gelatinase MMP-2 were the predominant active MMPs at both sites. Interestingly, the major MMP inhibitor TIMP-1 was...

Surface modification of poly([epsiv]-caprolactone) porous scaffolds using gelatin hydrogel as the tracheal replacement

24th July 2010

This study evaluates the feasibility of poly([epsiv]-caprolactone) as a tracheal replacement. To improve biocompatibility, the lumen was modified by gelatin hydrogel crosslinked with two different reagents, EDC and genipin. It was found that the choice of crosslinking agents could significantly affect human lung carcinoma cell proliferation. Genipin-crosslinked gelatin hydrogel had significantly better cell proliferation than EDC-crosslinked hydrogel. The study further investigated the performance of the PCL tube modified by genipin-crosslinked gelatin, using a rabbit tracheal implantation model with implants harvested and histologically examined. In vivo results showed that the PCL tube possessed suitable mechanical properties for resisting collapse during implantation. Additionally, PCL ...

Optimized electro- and wet-spinning techniques for the production of polymeric fibrous scaffolds loaded with bisphosphonate and hydroxyapatite

24th July 2010

This research activity was aimed at the development of composite bioactive scaffolds made of biodegradable three-arm branched-star poly([epsiv]-caprolactone) (*PCL), hydroxyapatite nanoparticles (HNPs) and clodronate (CD), a bisphosphonate that has demonstrated efficacy in the treatment of various bone diseases and as an anti-inflammatory drug. During the experimental work, the processing conditions for the fabrication of fibrous meshes, by either electrospinning or wet-spinning, were optimized. Stemming from a previous research activity on electrospinning of *PCL, *PCL/HNPs 3D meshes were developed, evaluating the influence of fabrication parameters on the fibres' morphology. By exploiting the binding affinity of bisphosphonates for hydroxyapatite, a methodology was set up for obtaining a...

Growth differentiation factor-5 regulation of extracellular matrix gene expression in murine tendon fibroblasts

22nd July 2010

This study was an attempt to identify potential mechanisms controlling the response of fibroblasts to injury and GDF-5, in the pursuit of improved tissue regeneration. There were two sets of experiments. Isolated mice Achilles tendon fibroblasts were treated with different concentrations of rGDF-5 (0-100 ng/ml) for 0-12 days in cell culture. The temporal effect of rGDF-5 on ECM gene expression was analysed for type I collagen and aggrecan expression. Microarray and gene expression analysis were performed on cells treated with 100 ng/ml for 4 days. Forty-five mice underwent bilateral mid-substance Achilles tendon tenotomy and suture repair. Repair sites were injected with 10 µg rGDF-5 or saline. Tendons were assessed histologically at 2, 4 and 6 weeks. Expression of ECM genes procollagen I...

Impact of pre-existing elastic matrix on TGF[beta]1 and HA oligomer-induced regenerative elastin repair by rat aortic smooth muscle cells

22nd July 2010

This study will help in customizing therapies for elastin regeneration within AAs, based on cause and location. Copyright © 2010 John Wiley & Sons, Ltd. (Source: Journal of Tissue Engineering and Regenerative Medicine)

In situ functionalization of wet-spun fibre meshes for bone tissue engineering

22nd July 2010

Bone tissue engineering success strongly depends on our ability to develop new materials combining osteoconductive, osteoinductive and osteogenic properties. Recent studies suggest that biomaterials incorporating silanol (Si[bond]OH) groups promote and maintain osteogenesis. The purpose of the present research work was to provide evidence that using wet-spinning technologies and a calcium silicate solution as a coagulation bath, it was possible to develop an in situ functionalization methodology to obtain 3D wet-spun fibre meshes with Si[bond]OH groups, through a simple, economic and reliable process. SPCL (blend of starch with polycaprolactone) fibre meshes were produced by wet-spinning, using a calcium silicate solution as a non-solvent and functionalized in situ with Si[bond]OH groups. ...

Three-dimensional culture of mouse bone marrow cells within a porous polymer scaffold: effects of oxygen concentration and stromal layer on expansion of haematopoietic progenitor cells

22nd July 2010

In conclusion, the 3D co-culture method of haematopoietic cells with stromal cells under hypoxic conditions was confirmed to be effective in expanding HPCs and erythroid cells, and this method seemed to be useful for developing an ex vivo expansion method for haematopoietic cells. Copyright © 2010 John Wiley & Sons, Ltd. (Source: Journal of Tissue Engineering and Regenerative Medicine)

Effectiveness factor and diffusion limitations in collagen gel modules containing HepG2 cells

22nd July 2010

A major obstacle in tissue engineering is overcoming hypoxia in thick, three-dimensional (3D) engineered tissues, which is caused by the diffusional limitations of oxygen and lack of internal vasculature to facilitate mass transfer. Modular tissue engineering is a bio-mimetic strategy that forms scalable, vascularized and uniform 3D constructs by assembling small (sub-mm), cell-containing modules. It was previously assumed that mass transfer resistance within the individual modules was negligible, due to their small size. In the present study, this assumption was tested using theoretical analysis of oxygen transport within the module (effectiveness factor) and experimental studies. Small (400 µm diameter post-contraction) and large (700 µm diameter post-contraction) HepG2-collagen module...

Scaffold-free 3D cellulose acetate membrane-based cultures form large cartilaginous constructs

22nd July 2010

Scaffold-free three-dimensional (3D) cultures provide clinical potential in cartilage regeneration. The purpose of this study was to characterize a scaffold-free 3D membrane-based culture system, in which human articular chondrocytes were cultivated on a cellulose acetate membrane filter, and compare it to pellet and monolayer cultures. Chondrocytes were expanded in monolayer culture for up to 5 passages, transferred to membrane-based or pellet cultures and harvested after 7 or 21 days. The chondrogenic potential was assessed by histology (toluidine blue, safranin-O), immunohistochemistry for collagen type II and quantitative analysis of collagen type II [alpha]1 (COL2A1). Membrane-based cultures (P1) formed flexible disc-like constructs (diameter 4000 µm, thickness 150 µm) with a large ...

Biological safety of human skin-derived stem cells after long-term in vitro culture

21st July 2010

The aim of this study was to investigate the biological safety of human skin-derived mesenchymal stem cells (HSMSCs) cultured in vitro by detecting changes in karyotype, major histocompatibility complex expression and tumorigenicity. Before the 21st passage of the in vitro culture, cell surface markers were analysed by flow cytometry; major histocompatibility complex expression was detected by RT-PCR and flow cytometry. The tumorigenicity of HSMSCs was tested using SCID mice and observing changes in the injection site and pathological sections. Flow cytometry demonstrated that HSMSCs express CD73, CD105 and vimentin, but haematopoietic markers CD34, CD45 and CD19 were not expressed. Human leukocyte antigen (HLA-I and HLA-DR) mRNA was detected by RT-PCR; the protein expression of HLA-I was ...

Shear-aggregated fibronectin with anti-adhesive properties

21st July 2010

Biomaterials based on proteins, such as fibronectin, have the potential to guide cell and tissue behaviour during healing as a function of their unique mechanical and bioactive properties. Fibronectin has been reported as a scaffold for attachment of fibroblasts and subsequent deposition of collagen. We have recently developed a derivative process of shear-aggregated fibronectin that prevents cell attachment without causing cell death. This has potential applications in clinical situations where adhesions form across gliding surfaces and cause loss of function, e.g. peritoneal or flexor tendon adhesions. This in vitro study tested this derivative fibronectin biomaterial and its effects on aggressive adhesion-forming cells, using rabbit flexor tendon synovial fibroblasts. We investigated de...

Chondrogenic differentiation of human mesenchymal stem cells in micro-masses is impaired by high doses of the chemokine CXCL7

21st July 2010

Chemokines have been shown to recruit human mesenchymal stem cells (MSCs) and are suggested to be promising candidates for in situ tissue engineering. The aim of our study was to analyse the effect of CXCL7, a chemokine that has the capacity to recruit MSCs, on the chondrogenic differentiation of MSCs. Bone marrow-derived MSCs were cultured in high-density micro-masses under serum-free conditions and were co-stimulated with 0-100 nM CXCL7 in the presence of 10 ng/ml transforming growth factor-[beta]3 (TGF[beta]3). Micro-masses stimulated without growth factors and chemokines served as controls. Histological staining of proteoglycan, immunostaining of type II collagen, staining of mineralized matrix according to von Kossa as well as real-time gene expression analysis of typical chondrogenic...

Cyclosporine-impregnated allograft bone sterilized with low-temperature plasma

21st July 2010

Deep-freezing, freeze-drying and gamma ([gamma])-irradiation have deleterious effects on bone healing and mechanical properties of allograft bones. We tried preparing bone allografts using cyclosporine plus low-temperature-plasma sterilization. To explore the feasibility of this method of preparation, segmental defects in the right radii of rabbits were repaired with cyclosporine-impregnated allograft bones (CABs) sterilized with low-temperature-plasma (in the study group) and deep-frozen/freeze-dried irradiated allograft bones (D/FIABs) (in the control group). X-ray and quantitative histological analysis, peripheral blood T lymphocyte subset analysis and CD25 molecule immunohistochemistry stain, the four-point bending test and safety evaluations were respectively conducted to compare bone...

Tissue-engineered constructs based on SPCL scaffolds cultured with goat marrow cells: functionality in femoral defects

5th July 2010

This study aims to assess the in vivo performance of cell-scaffold constructs composed of goat marrow stromal cells (GBMCs) and SPCL (a blend of starch with polycaprolactone) fibre mesh scaffolds at different stages of development, using an autologous model. GBMCs from iliac crests were seeded onto SPCL scaffolds and in vitro cultured for 1 and 7 days in osteogenic medium. After 1 and 7 days, the constructs were characterized for proliferation and initial osteoblastic expression by alkaline phosphatase (ALP) activity. Scanning electron microscopy analysis was performed to investigate cellular morphology and adhesion to SPCL scaffolds. Non-critical defects (diameter 6 mm, depth 3 mm) were drilled in the posterior femurs of four adult goats from which bone marrow and serum had been collected...

Developing a tissue-engineered model of the human bronchiole

4th July 2010

Scientists are always looking for new tools to better mimic human anatomy and physiology, especially to study chronic respiratory disease. Airway remodelling is a predominant feature in asthma and occurs in conjunction with chronic airway inflammation. Both the inflammatory and repair processes alter the airway wall which is marked by anatomical, physiological and functional changes. A tissue-engineered model of bronchiole remodelling presents a novel approach to investigating the initiation and progression of airway remodelling. By developing a unique bioreactor system, cylindrical-shaped bronchioles constructed from well-characterized human lung primary cells have been engineered and examined with a much greater control over experimental variables. We have grown human bronchioles compose...

Both sides nanopatterned tubular collagen scaffolds as tissue-engineered vascular grafts

4th July 2010

In this study, a novel tubular collagen scaffold possessing nanopatterns in the form of channels (with a 650 nm periodicity) on both sides was designed and examined after seeding and co-culturing with vascular cells. Initially, the exterior of the tube was seeded with human vascular smooth muscle cells (VSMCs), cultured for 14 days, and then human internal thoracic artery endothelial cells (HITAECs) were seeded on the inside of the tube and cultured for a further week. Microscopy revealed that nano-scale patterns could be reproduced on collagen with high fidelity and preserved during incubation in vitro. The VSMCs were circumferentially orientated with the help of these nanopatterns and formed multilayers on the exterior, while HITAECs formed a continuous layer on the interior, as is the c...

Using swept-source optical coherence tomography to monitor the formation of neo-epidermis in tissue-engineered skin

4th July 2010

There is an increasing need for a robust, simple to use, non-invasive imaging technology to follow tissue-engineered constructs as they develop. Our aim was to evaluate the use of swept-source optical coherence tomography (SS-OCT) to image tissue-engineered skin as it developed over several weeks. Tissue-engineered skin was produced using both de-epithelialized acellular dermis (DED) and amorphous collagen gels. In both cases the epidermis could be readily distinguished from the neodermis, based on a comparison with standard destructive histology of samples. Constructs produced with DED showed more epidermal/dermal maturation than those produced using collagen. The development of tissue-engineered skin based on DED was accurately monitored with SS-OCT over 3 weeks and confirmed with conven...